CD56 expression predicts response to Daratumumab-based regimens

International staging system

• Cell lines, reagents, Flow cytometry analysis, western blot analysis, RNA extraction and quantitative real-time PCR analysis, and transfections.• Immunophenotype for CD56 and CD38 clone size evaluation.
• Patient characteristics, definition of outcomes, and statistical analysis.
• Supplementary Table S2.Characteristics of n = 84 patients treated with Daratumumab in combination with immunomodulatory drugs (IMiDs).

Supplementary Figures:
• Supplementary Figure S1.Modulation of CD56 and its downstream targets by anti-CD38 monoclonal antibodies.
• Supplementary Figure S2.Outcomes of patients treated with Dara based on CD56 clone size.
• Supplementary Figure S3.Role of CD38 and CD56 correlation in the response to Dara.
• Supplementary Figure S4.CREB1 does not regulate CD38 expression in MM.
• Supplementary Figure S6.Role of CD56 and 1q+ status in the response to anti-CD38 monoclonal antibodies.
Western Blot Analysis: MM cells were harvested and lysed using RIPA lysis buffer (Cell The percentage of Daratumumab-mediated ADCC was calculated using the following formula: % lysis = (baseline cells-treated cells)/baseline cells*100.
Supplementary Table S4.Immune signature genes related to Fig. 2D and Fig. S5A.
Median PFS and 95% CI are reported in the insert of the plot.Log-rank p = 0.690.D. PFS from the first day of Dara single agent therapy in patients with less (n = 9, Low CD56-blue) or more than 10% of CD56-expressing MM clonal cells (n = 21, High CD56-fuchsia).Median PFS and 95% CI are reported in the insert of the plot.Log-rank p = 0.334.SupplementaryFigure S3.Role of CD38 and CD56 correlation in the response to Dara. A. Progression-free survival (PFS) from the first day of Dara-IMiD therapy in patients with Low CD38, Low CD56 (n = 14, blue), High CD38, Low CD56 (n = 10, black), Low CD38, High CD56 (n = 25, fuchsia), and High CD38, High CD56 (n = 35, orange).Median PFS and 95% confidence interval (CI) are reported in Table S3.Log-rank p = 0.002.B. Log2 CD38 expression values in patients with Low or High CD56 Log2 expression in the CoMMpass MMRF database.Total patients = 809, p < 0.0001 (****).Blue lines indicate median values.Dotted black lines indicate the 25 th and 75 th percentiles.C. Fold changes of CD56 mRNA levels in U266 control cells (CNT) or U266 cells overexpressing CD56 in the 4 replicates used in the manuscript.Ratio is normalized to the control cells.p = 0.0080 (**).D. Flow cytometry staining for CD56 to confirm overexpression of CD56 in U266 cells.E. Fold changes of CD56 mRNA levels in MM.1S control cells (CNT) or MM.1S cells overexpressing CD56 in the 2 replicates used in the manuscript.Ratio is normalized to the control cells.p = 0.038 (*).F. Fold changes of CD38 Mean Fluorescence Intensity (MFI) and mRNA levels in MM.1S control cells (CNT) or MM.1S cells overexpressing CD56.Ratio is normalized to the control cells.n = 2 replicates.MFI p = 0.04 (*); mRNA p = 0.028 (*).

Antibody-dependent cell-mediated cytotoxicity (ADCC) assay
signaling, Danvers, MA, United States, Cat.No. 9806), with addition of 1mM PMSF (Cell signaling, Cat.No. 8553).Cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose antibody (1: 5,000 dilution).All antibodies were prepared in milk 5% diluted in TBS-T(Biorad,   Hercules, CA, United States, Cat.No.1706435).RNA extraction and quantitative real-time PCR analysis: RNA was extracted using TRIzol procedure (Invitrogen, Life Technologies, Carlsbad, CA, United States).After quantification, 1,000-2,000 ng of RNA were used to synthesize cDNA by ProtoScript® II First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, United States) according to the manufacturer's instructions.To evaluate the expression levels of genes of interest (see table below), quantitative real-time PCR analysis was performed using SYBR GREEN PCR Master Mix (Applied Biosystems, CA, United States) after optimization of the primer conditions.Quantitative real-time PCR analysis was performed on a ViiA 7 Real-Time PCR System (Applied Biosystems, CA, United States).Data were analyzed using the ∆∆ Ct method.GAPDH was used

Table S1 .
This study was approved by the Ohio State University Institutional Review Board (OSU-23237) after providing written informed consent for the Ohio State University MM registry (OSU-10115) in accordance with the Declaration of Helsinki.Patients with a diagnosis of MM accordingly to the International Myeloma Working Group consensus criteria, who were treated at the Ohio State University Wexner Medical Center Comprehensive between January 1 st , 2016 and July 1 st , 2023 with a regimen containing Daratumumab-Dara (n = 152) or Isatuximab-Isa (n = 32) either as single agents or in combination with immunomodulatory drugs (IMiDs, lenalidomide or pomalidomide) or proteasome inhibitors (PIs, was calculated from the first day of therapy with Dara or Isa at last follow-up or death.PFS was estimated using the Kaplan-Meier method, using Log-rank test to compare the groups.Cox proportional hazard regression models were used to estimate the hazard ratios for risk of progression or death.The multivariable Cox model was built including all the variables significantly associated with PFS in the univariable analysis or variables known to affect outcomes in MM.Analyses were performed using IBM SPSS Statistics version 28, and all statistical tests were two-sided with statistical significance at 0.05.Characteristics of n = 152 patients treated with Daratumumab. expressing clonal MM cells (clone size) is routinely performed for clinical purposes on each bone marrow aspirate from MM patients in the Flow Cytometry Laboratory at The Ohio State University Wexner Medical Center, which is regulated under Clinical Laboratory Improvement Amendments.Specifically, flow cytometric analysis is performed using a ten-color technique with a gating strategy based on CD45 staining and light side scatter characteristics.Plasma cells are CD138 positive, kappa/lambda restricted.A cutoff of <10% or >10% of CD56-expressing was used to define Low or High CD56 subgrouping, while the median CD38 clone size value was used as cutoff to define Low or High CD38 subgrouping.Patient characteristics:bortezomib and carfilzomib) were included in the retrospective analysis.There were no exclusions based on age except for individuals younger than 18 years, since the Ohio State University Comprehensive Cancer Center Multiple Myeloma clinic is an adult oncology clinic.There were no exclusions based on gender, racial, or ethnic groups for the proposed research.1q+statuswasdefinedbasedon1q21copynumberspresent in at least 20% of the CD138 + enriched cells by FISH as: 1q+ negative (2 copies), gain(1q) (3 copies), and amplification(1q) (4 copies or more).CD138 enrichment was performed using the EasySep Human CD138 Positive Selection Cocktail (STEMCELL Technologies, Vancouver, Canada).Definition of outcomes and statistical analysis:Response criteria were based on the International Myeloma Working Group synopsis and included complete responses (CR), very good partial responses (VGPR), partial responses (PR), minimal responses (MR), stable disease (SD), or progressive disease (PD).Demographic and disease characteristics were summarized using medians and ranges for continuous variables, and frequencies and percentages for categorical variables and compared using chi-square test or Fisher's exact test.Primary endpoints were progression-free survival (PFS) from first day of Dara or Isa-based regimens to progression or death censoring the patients without progression at last follow-up.Overall survival (OS) (ENSG00000004468) FKPM levels were normalized to Log2 values and correlated as dependent variables in the linear regression analysis, or with 1q21+ status.For Gene set enrichment analysis (GSEA), patients were divided based on median CD56 levels.GSEA was used to score enrichment levels and significance of a predefined immune signature reported in TableS4and derived from Chen et al(Ref.8).Volcano plot analysis was used to show differences in expression and adjusted p-values.SupplementaryAbbreviations: n, number; MM, Multiple Myeloma; p, p-value; NHW, non-Hispanic white; NHB, non-Hispanic black; LC, light chain disease; ISS, International staging system; IMiDs, immunomodulatory drugs; PIs, proteasome inhibitors; CR, complete response; VGPR, very good partial response; PR, partial response; MR, minimal response.

Table S2 .
Characteristics of n = 84 patients treated with Daratumumab in combination with immunomodulatory drugs (IMiDs).

Table S5 .
Characteristics of n = 32 patients treated with Isatuximab.CI) are reported in the insert of the plot.Log-rank p = 0.322.C. PFS from the first day of Dara-proteasome inhibitor (PI) therapy in patients with less (n = 15, Low CD56blue) or more than 10% of CD56-expressing MM clonal cells (n = 23, High CD56-fuchsia).